1. Field of the Invention
The present invention relates to materials and methods for rapid detection and characterization of nucleic acid sequences and sequence variability. The present invention relates to means for changing the separation mobility of single stranded nucleic acids in a temperature dependent manner. Changes in separation mobility are used to detect known and unknown single base changes in nucleic acids.
2. Background Art
Genetic variability observed between specimens of any species is the result of polymorphism developed during biological evolution and from spontaneous mutations accumulated during the lifespan of individuals. Establishing the correlation between genetic variations, the environment, and the phenotype of an organism (or a population) could provide vital information for understanding the basic mechanism of life.
One of the most often observed polymorphisms is a Single Nucleotide Polymorphism (SNP) where a single base pair distinction exists in the sequence of a nucleic acid stranded compared to the most prevalently found (wild type) nucleic acid strand. When a SNP occurs in the gene coding for a structural protein or its regulating region, the polymorphic locus may influence the function of the encoded protein and cells which express the protein, in addition to affecting the organismal phenotype.
In most cases, the observed phenotype in higher organisms is the result of the interaction between several gene products. The phenotype resulting from a mutation or SNP at a first locus may be offset, or compensated for by mutations at a second locus. As the compensation/adjustment mechanism influences the final phenotype, the polymorphic pattern and mutation frequency correlating to any phenotype might only be slightly different in an affected group compared to a control group. To establish such correlations, genome-population-wide association studies are usually required.
Since such studies could significantly improve the quality of human life, for example, by introducing DNA-based diagnostics and pharmacogenomics practice into hospital routines, establishing a reliable methodology of DNA mutation and polymorphism analysis on the genome-population-wide scale is strongly needed. Despite the fact that sequencing of DNA becomes more and more a routine methodology, none of the currently existing technical methods for genetic sequence variation detection could be applied to, e.g., human genome-population-wide genetic surveys in an acceptable time and cost scale.
At present, at least three steps can be identified for the mutation/SNP detection process. In the first step, the regions/genes of the genome to be analyzed must first be selected. In the second step, screening for the presence of mutations/SNP in the selected genes/regions is conducted. In the last step sequencing of selected samples may be performed. The accuracy of the mutation/SNPs detection process depends on the amount of target nucleic acid (NA) in the analyzed sample. Several standard methods are available for purifying nucleic acids from the starting material, in addition to the wide selection of commercial kits available for nucleic acid purification.
When a sufficient amount of the isolated target nucleic acid is acquired, “direct mutation detection” methods can be applied to detect nucleic acid. Methods for “direct detection” of specific sequences in nucleic acids are mostly based on the NA/NA hybridization (e.g, Branched DNA method bDNA-Urdea et al., Gene 61:253–264 (1987) or protein/NA interaction (Restriction Fragment Length Polymorphism-RFLP)).
However, when the amount of the target nucleic acid in the analyzed sample is too low for direct analysis, an amplification step of selected nucleic acid fragments is necessary. The most popular method used for the amplification of target nucleic acid fragments is Polymerase Chain Reaction (PCR) as described in U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis et al. The process comprises treating separate complementary strands of the target nucleic acid with a molar excess of two oligonucleotide primers; extending the primers with thermostable DNA polymerase to form complementary primer extension products which act as templates for synthesizing the desired nucleic acid sequence; and detecting the amplified sequence. The steps of the reaction may be carried out stepwise, or simultaneously, and can be repeated as often as desired. Other methods used for nucleic acid amplification include:                Ligase Chain Reaction (LCR or LAR)—described by Barany, Proc. Natl. Acad. Sci. 88:189 (1991). In LCR, four oligonucleotides, two adjacent oligonucleotides which uniquely hybridize to one strand of target DNA, and a complementary set of adjacent oligonucleotides, which hybridize to the opposite strand are mixed and DNA ligase is added to the mixture. Provided that there is complete complementarity at the junction ligate will covalently link each set of hybridized molecules. Importantly, in LCR, two probes are ligated together only when they base pair with sequences in the target sample without gaps or mismatches. Repeated cycles of denaturation, hybridization, and ligation amplify a short segment of DNA. Because the four oligonucleotides used in this assay can pair to form two short ligatable fragments, there is the potential for the generation of a target-independent background signal. Another limitation is that the method can only be used for the detection of known genetic variations.        
Self-Sustained Synthetic Reaction (3SR/NASBA)—described by Guatelli et al., Proc. Natl. Acad. Sci. 87:1874–1878 (1990) is a transcription-based nucleic acid amplification method that can exponentially amplify 200–300 base pair long RNA sequences at a uniform temperature.
With amplified target nucleic acid, several methods can be used for subsequent mutation and polymorphism detection and characterization. Such methods might be divided into two groups:                biological or specific, for example, RFLP, hybridization, Allele Specific Amplification (ASO) PCR, pyrosequencing, etc.        physical-chemical scanning methods, for example, SSCP, Denaturing Gradient Gel Electrophoresis (DGGE),Denaturing High Performance Liquid Chromatography DHPLC, cleavage based methods, etc.        
Comprehensive reviews of techniques used for SNP/mutation detection are described in Electrophoresis No. 6/99, vol. 20 and U.S. Pat. No. 5,719,028.
Biological or specific methods are based on the recognition of a specific nucleic acid sequence, therefore they are primarily suitable for the detection of a known SNP/mutation. In contrast, the physical or scanning methods require no prior knowledge of investigated sequences and are used for detecting and/or identifying any SNP/point mutations. Thus, these methods can be used for such applications as screening of highly variable genetic regions.
An example of a biological method for detecting genetic diversity is competitive PCR, described in U.S. Pat. No. 5,582,989. In that method, two different sets of primer pairs are used to amplify a target nucleic acid sequence. According to this method, one set of primers recognizes the wild type sequence and the other set recognizes sequence containing the selected point mutation. Another example is a method used for detecting the specific SNPs presence in the target nucleic acid based on the Allele Specific Amplification (ASO) as described by Shuber (1997) in U.S. Pat. No. 5,633,134. Unfortunately, PCR reactions may generate false results due to amplification of nucleic acid sequences to which the primers are not perfectly complementary. Accordingly, depending on the reaction conditions (temperature, ionic strength) either set of competitive primers can prime elongation of either the wild type or the mutant sequence.
Another group of techniques developed for SNP/mutation detection and analysis is based on the hybridization properties of the NA (biochips), see, e.g., Sapolsky et al. (1999), U.S. Pat. No. 5,858,659; Nerenberger, M., et al. (2000), WO 0061805; Arnold, L., et al. (2000), WO 0050869. Also, the RFLP method mentioned above can be used for detecting genetic variability in amplified nucleic acid fragments.
However, with any of the techniques described above the nature of the suspected genetic variability must be known prior to testing. Thus, those techniques are inapplicable when one needs to detect the presence of a mutation/polymorphism of an unknown character and position.
Only the physical-chemical methods are capable of detecting either known, or unknown mutations, and polymorphisms in any selected genome of interest.
One of these physical-chemical methods called Denaturing Gradient Gel Electrophoresis (DGGE), is based on changes in electrophoretic mobility of analyzed NA/NA hybrids when subject to denaturing conditions. In the DGGE method, genetic variants can be distinguished based on the differences in melting properties of NA homoduplexes versus heteroduplexes, sometimes due to differences in a single nucleotide which results in changes in electrophoretic mobility. To increase the number of mutations that can be recognized by DGGE, nucleic acid fragments amplified by PCR reaction are “clamped” at one end by a long stretch of G-C base pairs (30–80) to avoid complete dissociation of the strands while allowing for complete denaturation of the sequence of interest (Abrams et al., Genomics 7:463–475 (1990); Sheffield et al., Proc. Natl, Acad. Sci., 86:232–236 (1989); and Lerman and Silverstein, Meth. Enzymol. 155:482–501 (1987)). To increase the SNP/mutation detection rate, a temperature gradient is introduced during the separation of amplified NA target (Wartell et al., Nucl. Acids Res. 18:2699–2701 (1990)).
Some limitations of the DGGE method arise from the fact that the denaturing conditions (temperature and urea concentration) depend on the analyzed sequence, so there is a need for optimization of the denaturing conditions for each target sequence. Reproducibility of the method depends on the accurate gradient gel preparation and precise gel temperature control. Extra expense is often incurred from having to synthesize a GC clamping tail for each sequence to be tested. Further, the time required to analyze each sample is often quite lengthy.
One of the most widely used physical-chemical methods for SNPs/mutation detection and identification is Single Strand Conformation Polymorphism (SSCP). With SSCP analysis, single base differences in single stranded NA fragments are recognized by differences in their mobility during separation under native conditions (Orita et al., Genomics 5:874–879 (1989)). In native conditions the single stranded NA fragments adopt a secondary structure according to their sequence and actual physical conditions. Since the electrophoretic mobility of a single stranded nucleic acid molecule depends on its net electric charge and three-dimensional (3-D) conformation, modification of any single nucleotide in the DNA fragment can result in a different 3-D conformation and thus influence the separation mobility. In some cases, single nucleotide modifications, however, result in different 3-D conformation only under particular physical-chemical conditions, of which the most important are ionic strength, pH and temperature. Based on variables such as temperature and ionic strength, the number and energetic stability of 2-D conformers of any single stranded nucleic acid molecule can be calculated using the nearest-neighbor thermodynamics algorithm which is known to those skilled in the art and is available on-line at http://bioinfo.math.rpi.edu/˜mfold/dna/. The presence of energetically stable 2-D conformers is predictive of the formation of stable 3-D spatial conformers.
Despite the simplicity of administering the SSCP method and relatively high sensitivity (above 90%), some limitations of the SSCP method have been reported. The most important limitation is the difficulty in obtaining consistent results. This is caused by the lack of a theoretical background from which predictions of the optimal separation conditions may be based (ionic strength, pH, temperature) for a particular NA fragment. The observed variability of SSCP results, according to many authors, results from the inconsistency of the separation conditions and on the mutation location within the analyzed NA fragment (Glavac and Dean, 1993, Hayashi and Yandell, 1993, Liu and Sommer, 1944). For example, to lower the temperature influence on the electrophoretic separation, low voltage power has been applied, however this has resulted in lengthy times of separation of up to 12–14 hours. Sensitivity of the SSCP method could be increased to close to 100% by applying at least two different separation conditions for the analysis of the same target nucleic acids. The separation conditions which are most influential on the separation mobility of single stranded nucleic acids are the type of porous support, the chemical composition of the separation buffer and temperature (Liu et al. in WO0020853). However, the time and cost of such an approach rise proportionally to the number of additional separations applied to analyze the same set of samples and would be quite difficult to use in routine diagnostics.
Genetic variability can also be determined based on a special mass-spectroscopy-analysis of the target NA by Monforte (1998) WO98/12355, Turano et al. (1998) WO98/114616 and Ross et al. (1997) Anal Chem. 15:4197–4202. This method requires quite expensive and specialized equipment, which is a major consideration and drawback.
In summary, there is a strong need for analytical tools and methods that would allow for reliable, time and cost-effective and close to 100% detection of genetic variability in nucleic acids. To become a useful diagnostic or technological tool, it should operate in full automatic mode with the capability of analyzing several samples at the same time. Such tools and methods would allow for more widespread diagnostic screening of humans with predispositions for various life treating diseases than is currently possible which could result in changing the health care paradigm from diagnosis and treatment to disease prevention.